In severe or chronic cases the disease of Diabetes is usually treated with injection preparations containing insulin, e.g. porcine insulin, bovine insulin or human insulin.
A number of different processes for the biosynthetic production of human insulin are known. Common to all of them is that the DNA strand coding for either the entire proinsulin, a modified form hereof or for the A and B chain separately is inserted into a replicable plasmid containing a suitable promoter. By transforming this system into a given host organism a product can be produced which can be converted into authentic human insulin in a manner known per se, cf. e.g. EP B1 85,083 or EP B1 88,117.
Some known processes for biosynthesis of proinsulin or similar insulin precursors and there conversion into insulin are described below.
Proinsulin may be prepared biosynthetically by using the method disclosed in the specification of European patent application No. 121,884. In this method the gene coding for proinsulin is inserted into a yeast strain and after culturing such transformed yeast strain proinsulin can be isolated from the culture medium. Hereafter, proinsulin can be converted into insulin in a manner known per se. Yields of proinsulin obtained by this method are, however, unsatisfactory low for commercial production.
Insulin precursors of the formula B-X-A wherein B and A represent the B and A chain, respectively, of human insulin and X represents a polypeptide comprising at least 2 amino acid residues, preferably from 6 to 35 amino acid residues, are known from the specification of Danish patent application No. 5284/87. The precursors can be enzymatically digested into human insulin by treatment with trypsin and carboxypeptidase B in the presence of certain metal ions.
European patent application No. 195,691 discloses closely related insulin precursors of the formula B-X-Y-A wherein B and A represent the B and A chain, respectively, of human insulin, cross-linked through sulphur bridges as in human insulin, and X and Y each represents a lysine or arginine residue, as well as the preparation of said precursors. These precursors can be enzymatically digested into human insulin by treatment with trypsin and carboxypeptidase B. Moreover, said precursors can undergo tryptic digestion into des-B30-insulin; however, a considerable amount of A.sub.o Arg-des(B30)-insulin is formed which only slowly undergoes further digestion.
It is an object of the invention to provide novel insulin precursors which are generated in high yields in yeast and which furthermore can be converted into human insulin or insulin analogues with minimal formation of undesired by-products.
The insulin precursors of the invention are characterized by the following amino acid sequence EQU B(1-29)-X.sub.1 -X.sub.2 -Y.sub.2 -Y.sub.1 -A(1-21)
wherein B(1-29) are the 29 first amino acid residues of the B chain of human insulin starting from the N-terminus, A(1-21) are the 21 amino acid residues of the A chain of human insulin, X.sub.1 represents a peptide bond or one or more arbitrary amino acid residues, X.sub.2 represents Glu or Asp, and Y.sub.1 and Y.sub.2 each represents Lys or Arg, the positions A6 and A11, A7 and B7, and A20 and B19, respectively, are connected through sulphur bridges, and, if desired, one or more of the amino acid residues of the chains B(1-29) and A(1-21) are substituted by another amino acid residue.
The invention is based on the surprising recognition that the above insulin precursors are either generated in an extremely high yield as compared with the compound B-Lys-Arg-A known from European patent application No. 121,884 or that when these precursors are digested by trypsin des-B(30)-insulin is obtained in high yields with no or very little formation of A.sub.o -Arg-des-B(30)-insulin and A.sub.o -Lys-des-B(30)insulin or both.
It is preferred that X.sub.1 represents a peptid bond or one amino acid residue.
Preferred precursors of the invention are represented by the formulas B(1-29)-Asp-Lys-Arg-A(1-21) and B(1-29)-Glu-Lys-Arg-A(1-21).
Des-B(30)-insulin can be converted into e.g. human insulin by enzymatically catalysed semisynthetic processes in a manner known per se.
The precursors may also be converted into human insulin by the transpeptidation method e.g. as described in U.S. Pat. No. 4,343,898.
Insulins in which one or more of the amino acid residues in the B(1-29) and A(1-21) chains are substituted by another amino acid residue may e.g. be the insulin derivatives showing protracted action and disclosed in the specification of international patent application WO 86/05497. In said insulin derivatives showing protracted action one or more of the amino acid residues of positions A4, A17, B13 and B21 are substituted by an amino acid residue having an uncharged side chain, e.g. an alkyl ester or an amid. Further insulin analogues which can be prepared according to the present invention are such insulins as described in European patent application Nos. 194,864 and 214,826.
The insulin precursors of the invention can be prepared by expressing a DNA sequence encoding an insulin precursor of the invention in a suitable expression system, preferably a yeast expression system.
The DNA sequence encoding the insulin precursors of the invention can be prepared from a DNA sequence encoding an insulin precursor B(1-30)-Lys-Arg-A(1-21) by in vitro mutagenesis or by oligonucleotide synthesis of the entire DNA sequence.
The invention is also related to a process in which a yeast strain transformed by a replicable expression vehicle comprising a DNA sequence encoding the insulin precursor with the above formula is cultured in a suitable culture medium, and then the precursor thus formed, optionally after isolation thereof, is converted into des-B(30)-insulin by tryptic digestion.
The present invention is furthermore related to a method for the preparation of human insulin or insulin analogues by which method a yeast strain transformed with a replicable expression vehicle comprising a DNA sequence encoding an insulin precursor of the above formula is cultured in a suitable culture medium whereupon the precursor thus formed is converted into human insulin or insulin analogues by known means.
To achieve secretion to the culture medium, the DNA sequence encoding the insulin precursors can be fused to another DNA sequence encoding a signal peptide functional in yeast. Secretion can be achieved by insertion in the expression vehicle of the yeast MF.alpha.l-leader sequence (Kurjan & Herskowitz, Cell 30, 933-943, 1982) or parts thereof. A preferred construction uses the DNA sequence encoding the entire MF.alpha.l-leader sequence including the dibasic site LysArg but excluding Glu-Ala-Glu-Ala which is the substrate for the yeast protease DPAP (dipeptidyl aminopeptidase). In that way, an efficient secretion of insulin precursors having the correct N-terminal is achieved. Other suitable leader sequences are synthetic yeast leader peptides as described in WO 89/02463.
The expression of the desired DNA sequence is under the control of a DNA sequence which is a promoter for transcription correctly positioned in relation to the DNA sequence being expressed. In the preferred embodiment the GAPDH (glyceraldehyd-3-phosphate-dehydrogenase) promoter is used. As the terminator of the transcription the terminator sequence of the MF.alpha.- l-gene is used.